There is compelling evidence that tumor angiogenesis is necessary for a tumor to grow beyond a minimal size. Tumor cell secreted angiogenic peptides induce the growth of new blood vessels into tumors. The stimulus for the production of these angiogenic factors is tumor hypoxia and it is accentuated by mutations in specific oncogenes and tumor suppressor genes. The Hypoxia Inducible transcription Factor (HIF) is a central regulator of this cellular response to hypoxia and an upstream activator of several angiogenic factors. A prototypic disorder in which HIF is deregulated is Von HippeI-Lindau (VHL) disease. Loss-of-VHL function directly stabilizes HIF and results in the transactivation of genes contributing to the malignant phenotype by promoting tumor cell proliferation. HIF may be necessary for growth of VHL+/+ and VHL-/- solid tumors. It is, therefore, conceivable that inhibiting HIF, instead of targeting each specific angiogenic factor separately, may be a powerful strategy to inhibit and/or prevent tumor formation. In Aim 1 we propose to generate stable and inducible clones derived from tumor cell lines, in which HIF activity is inhibited through expression of short interfering RNA. As a second strategy (Aims 1 and 2) we will use small molecule HIF inhibitors that we discovered in a high throughput screen. We will determine the molecular mechanism of inhibition and the effect of these compounds on the overall pro-angiogenic profile of VHL+/+ and VHL-/- tumor cell lines. Pharmacologically guided dosing regimens will be used to study the effect of inhibitors on xenografted and spontaneously developing tumors in mice. These studies will test whether HIF is necessary for tumor angiogenesis. Reagents generated at the course of these proof-of-principle experiments may provide the direct basis for anti-cancer drug development.